Cloning "Long Range" PCR products

Mike Dalrymple dalrymple at pplros.demon.co.uk
Mon Jul 24 07:02:03 EST 1995


Hello,

does anyone have experience of cloning these "extra-long" PCR products? We
have been trying to clone 8, 9 and 16Kbp fragments for some weeks now
with little
or no success. We have tried the usual things, e.g.: pGEM-T, blunt
cloning into
pBSIISK and pUC19, cloning into lambda (desperation!!) and most recently
the positive selection vector pZERO-1. We have varied insert/vector
ratios,
ligation volumes, changed ligase supplier, etc, etc. All our controls are
as
expected, i.e. the vector can accept insert, without insert the
background is
low. There is no reason to believe that these bits of DNA are unclonable
as others have done so by standard cloning techniques (OK I hear you say,
ask them for
the clone). The ability to PCR and clone large bits of DNA is fundamental
to a couple 
of projects we have on the go and so its important that we get the
technology to WORK.

Is there something funny about the ends of these things? Suggestions on an
email card to....

Mike Dalrymple



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